In the industrial production of polypeptides it is of interest to achieve a product yield as high as possible and to be able to control the said expression. One way to increase the yield is to increase the copy number of a gene encoding a polypeptide of interest. This can be done by placing the gene on a high copy number plasmid. However, plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells. Another way to increase the copy number of the gene of interest is to integrate it into the host cell chromosome in multiple copies. It has previously been described how to integrate a gene into the chromosome by double homologous recombination without using antibiotic markers (Hone et al., Microbial Pathogenesis, 1988, 5: 407-418); integration of two genes has also been described (Novo Nordisk: WO 91/09129 and WO 94/14968). A problem associated with integrating several copies of a gene into the chromosome of a host cell is instability of the multiple copies of the gene. Due to the sequence identity of the copies there is a high tendency for them to recombine and cross out of the chromosome during cultivation of the host cell unless a selective marker or other essential DNA is included between the copies, and selective pressure is applied during cultivation. This is especially true if the genes are located in relative close vicinity of each other. It has been described how to integrate two genes closely spaced in anti-parallel tandem to achieve better stability (Novo Nordisk: WO 99/41358).
In the above described solution to the stability problem each copy of the gene of interest requires its own promoter. In the case where several copies of a gene are integrated on the chromosome in different locations a variation in the level of expression might occur due to the different environment of the promoter. One way of obtaining high uniform levels of expression from multiple copies of a gene would be to transcribe all the copies from a polycistronic messenger controlled by one promoter region. This, however, would lead to the described instability of the construct.
It is therefore desirable to be able to integrate several copies of the same gene in close proximity to each other and being controlled by the same promoter in front of the first copy. Such a construct will normally be unstable when integrated on the chromosome due to the above described recombination taking place. It is therefore desirable to have alternative solutions to the stability problem experienced when having multiple copies of the same gene in close proximity on the host chromosome.
The present invention provides a solution in which at least two copies of the gene can be integrated in the same orientation on the host cell chromosome, either in different positions or in the same position e.g. as a tandem repeat.